mohl's half leaf experiment

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Experiments on Photosynthesis for High School

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The below mentioned article includes a collection of ten experiments on photosynthesis for high school.

1. Experiment to demonstrate Moll’s half-leaf experiment for showing that CO 2 , light, chlorophyll and water are necessary requirements for photosynthesis:

Requirements:

A potted plant, caustic potash, wide- mouthed bottle, iodine, split cork, water.

1. De-starch a potted plant by putting it in complete darkness for two days.

2. Fill partly a wide-mouthed bottle with strong solution of caustic potash and fit a split cork on its mouth.

3. Insert about half of the portion of a leaf of the de-starched plant into the bottle through the split cork (Fig. 36).

4. Place the whole apparatus in light after applying grease on the upper portion of split cork, and test the leaf for stach after about 10 hours.

Observations:

Portions of the leaf inside the bottle as well as in between the split cork show negative test for starch indicating the absence of photosynthesis while the portions outside the split cork show positive test for starch indicating the presence of process of photosynthesis in this region.

Negative starch test by the leaf portion present inside the bottle indicates that process of photosynthesis is absent in this region. This portion of leaf is getting all the essential requirements, i.e., light, chlorophyll and water except the CO 2 because the latter is absorbed by the caustic potash. Thus, it can be concluded that CO 2 is necessary for this process.

Negative test of starch, which is also shown by the portion of the leaf present in between the split of the split cork, can be explained that it is due to the lack of CO 2 and light, thus indicating that both of them are essential requirements.

Positive test of starch shown by the portions of the leaf present outside the bottle indicates that photosynthesis process is continuously going on there because all the essential requirements, i.e., light, chlorophyll, water and CO 2 are readily available to this portion.

That the chlorophyll is also an essential requirement for photosynthesis can be shown by testing starch in a variegated leaf. Only green portions of the leaf show positive starch test.

2. Experiment to demonstrate that oxygen is evolved during the process of photosynthesis:

Beaker, water, test tube, funnel, Hydrilla plant.

1. Fill the beaker with the water and take an aquatic plant, such as Hydrilla, in the beaker.

2. Cut the bases of the plants, tie them with a thread and cover them with an inverted funnel in such a fashion that the cut ends of plants are towards the neck of the funnel (Fig. 37).

3. Fill a test tube with the water and invert it on the upper end of the funnel.

4. Keep the whole apparatus in sunlight and observe for some time.

From the cut ends of the plant some bubbles are coming out continuously and they are collected at the top of the test tube by displacing the water. On testing this gas it is found that it is oxygen.

The liberated gas is oxygen and it is evolved due to the photolysis of water under the process of photosynthesis. The liberated gas comes in the intercellular spaces and ultimately evolves out through the stomata.

3. Experiment to compare the rate of photosynthesis under different conditions with the help of Wilmott’s bubbler:

Wilmott’s bubbler, water, Hydrilla, vaseline, papers of red, blue and green colours, heater, sodium bicarbonate, thermometer, etc.

1. Fill a Wilmott’s bubbler with pond water.

2. Cut the bases of the Hydrilla plants, tie them with a thread and insert them in the narrow tube of the bubbler in such a fashion that their cut ends are towards the upper side as shown in the Fig. 38.

3. Add some definite quantity of sodium bicarbonate in the water and note the number of bubbles coming out in definite time.

4. Increase a definite quantity of sodium bicarbonate with definite interval and note the increase or decrease in the number of bubbles.

5. Again fix up the apparatus in the same way as discussed above. But instead of adding sodium bicarbonate, keep the whole apparatus in sunlight and shade with definite intervals and note the number of bubbles in a definite time.

6. Fix up the apparatus afresh and now cover the bubbler with red paper and note the number of bubbles in definite time. Take also the readings of bubbles covering the bubbler with green and blue coloured papers in a definite time. Again fix up the apparatus afresh and now instead of adding any substance or covering the bubbler with coloured papers, keep it near the electric heater. Note the readings in different temperatures.

Arrange all readings of different conditions in the form of tables as follows:

Table I indicates that by adding sodium bicarbonate in the water the number of bubbles increases. This indicates that photosynthesis increases. Sodium bicarbonate is added for increasing the amount of CO 2 in the water and so it can be concluded that the rate of photosynthesis increases by increasing the quantity of CO 2 , but only till the light or some other factor starts to act as a limiting factor.

Observations of the Table II Indicate that the number of bubbles is more in sunlight than in shade, and so it can be said that photosynthesis is more in sunlight in comparison to shade.

Table III shows that the photosynthesis is highest in red light while lowest in green light.

With the observations of Table IV, it can be concluded that the rate of photosynthesis increases by increasing the temperature. Too much increase in temperature will show negative effects on photosynthesis and ultimately the plant will die in high temperature.

4. Experiment to show the effect of different wavelengths of light during the process of photosynthesis:

A large ‘Ganong’s light screen’-like box in which the leaf can be inserted, glass top covered with blue, green and red colours, plant twig, stand, iodine, etc.

1. Place a potted plant in darkness for about 24 hours. It will make its leaves de-starched.

2. Fix a de-starched leaf below the glass top of the box and keep the apparatus in sunlight (Fig. 39).

3. Detach the leaf after a few hours. The chlorophyll is removed.

4. Stain the leaf with iodine to test for the presence of starch.

5. Compare the intensity of starch in the three parts of the leaf.

The leaf part receiving green light shows negative staining for the starch.

The leaf part receiving red light is darkly-stained while that receiving blue light is next in the order.

1. Negative staining in the green region indicates that photosynthesis process has not taken place in this region. So, green wavelength is ineffective in photosynthesis.

2. Darkest staining in the red region indicates that maximum photosynthesis has taken place in this region. And this has finally resulted in the largest accumulation of starch in this region.

3. Second darkly-stained region is the blue region of the leaf. This indicates that photosynthesis has taken place in this region also, but it happened at a lower rate than that of red region.

So, red wavelength is most effective, the blue wavelength comes next in order and the green is least effective.

5. Experiment to determine the amount of chlorophyll ‘a’, chlorophyll ‘b’ and total chlorophyll in a given plant tissue:

Fresh green plant material (e.g., spinach leaves), mortar, pestle, 80% acetone, centrifuge.

The amount of chl ‘a’, chl ‘b’ and total chlorophyll is determined by the under mentioned method proposed by Anderson and Boardman (1964):

1. Take known amount of fresh green plant material, crush it and dissolve it in 80% acetone with the help of a mortar and pestle.

2. Centrifuge the samples of the so-formed pulpy material and take the supernatant.

3. Make the final volume of each sample to 5 ml with the help of 80% acetone.

4. Record the optical density (O.D.) for each sample at two wavelengths, i.e., 663 nm and 645 nm.

Calculations and results:

Amounts of chlorophyll ‘a’, chlorophyll ‘b’ and total chlorophyll are calculated according to the following formulae:

The observations indicate that the iodine test is positive only in the regions of the leaf which were exposed to sunlight (P) while on the other hand the unexposed regions show negative iodine test for starch. Because the ultimate product of photosynthesis is starch, hence it can be concluded that it is formed only in those regions which remain exposed to sunlight and not in others. So, light is essential for photosynthesis.

Related Articles:

• Experiment to Prove Light is Essential for Photosynthesis (With Pictures )
• Experiments on Carbohydrates | Biochemistry

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Early Experiments to Understand Photosynthesis: Historical Account of Photosynthesis

Early Experiments to Understand Photosynthesis: Have you heard the term photosynthesis? Have you ever wondered how we came to know that plants require sunlight or water for photosynthesis? Let us understand all these facts. Only photosynthesis is the means by which certain organisms can make their own organic food from inorganic raw material with the help of solar energy. The organisms performing photosynthesis are therefore called autotrophs. The study on photosynthesis started around 300 years ago. Simple experiments have shown that chlorophyll (green pigment of the leaf), light, and CO 2 are required for photosynthesis to occur.

Photosynthesis

Photosynthesis is the process of the plant by which they utilize sunlight, water (H₂O), carbon dioxide (CO₂), and chlorophyll molecules to form oxygen and glucose (C₆H₁₂O₆) molecules. It converts light energy into chemical energy. Let us study the various historical experiments of photosynthesis.

Learn Everything About Photosynthesis Here

Historical Account of Experiments on Photosynthesis

There have been several simple experiments done that led to a gradual development in our understanding of photosynthesis

i. Joseph Priestley (1733-1804):  In 1770, Priestly revealed the essential role of air in the growth of green plants through several experiments. He discovered oxygen in 1774. In an experiment done, Priestley observed that a candle is burning in a closed space, i.e., a bell jar, soon gets extinguished. Similarly, a mouse would die of suffocation in a closed space due to the unavailability of oxygen. Through his experiments, he concluded that both the burning candle and the mouse contaminates the air they use. But, when a mint plant was placed in the same bell jar, the mouse stayed alive, and the candle continued to burn. As a result, Priestley concluded that plants add to the air what a breathing mouse and burning candle remove or use.

ii. Jan Ingenhousz (1730-1799): Ingenhousz, through his experiments, showed that sunlight is essential for the plant process that helps to somehow purify the air fouled by the breathing of mice and the burning candle. In another experiment with an aquatic plant ( Hydrilla ), he showed that small bubbles were formed around the green parts of the plant in bright sunlight. While in the dark, no bubbles were formed. He identified those bubbles to be oxygen. Therefore,  he showed that in the presence of sunlight, only the green parts of the plants could release oxygen.

iii. Julius von Sachs (1854): He found that the green parts in plants are the place where glucose is made, and glucose is usually stored as starch. Later, he showed that the green substance in plants (now called chlorophyll) is located in special bodies (now called chloroplasts) within the plant cells. 

iv. T.W. Engelmann (1843-1909): He experimented on Cladophora using a prism; he split light into its spectral components and then illuminated a green alga kept with aerobic bacteria. The bacteria were used to detect the sites of oxygen evolution. He found that the bacteria accumulated mainly in the blue and red light regions of the split spectrum. And thus, the first action spectrum of photosynthesis was described. The empirical equation representing the total process of photosynthesis for organisms evolving oxygen was understood as

v. Cornelius van Niel (1897-1985): Van Neil, based on his studies of purple and green sulphur bacteria, demonstrated that during photosynthesis, oxygen evolved by the green plants comes from water and not from carbon dioxide. The hypothesis was later proved by using radioisotopic techniques.

Where H 2 A is the oxidisable compound (H 2 O or H 2 S).

The correct equation to represent the overall process of photosynthesis could thus be summed as

Where C 6 H 12 O 6 is glucose and O 2 is released from water.

vi. Ruben, Kamen, and Hassid used a heavy, but non-radioactive, stable isotope of oxygen 18 O to prove that O 2 evolved during light reaction comes from H 2 O and not from CO 2 .

Some Important Experiments Related to Photosynthesis

Let us now study certain experiments to study the need for light, chlorophyll and carbon dioxide for photosynthesis.

Experiment to Demonstrate that Light and Chlorophyll is Necessary for Photosynthesis: 

Aim: To demonstrate the use of light and chlorophyll in photosynthesis.

Materials Required: Destarched leaf, Black strip, Iodine solution.

Procedure: 

• Take a destarched potted plant having variegated leaves and cover 2-3 leaves with black paper. 
• Expose the potted plant to sunlight for 1-2 hours. 
• Pluck one covered leaf and one exposed leaf.
• Both the leaves are then dipped in iodine solution. 

Observation: The leaf which was covered does not pass the starch test proving that in the absence of light, photosynthesis cannot occur. The exposed leaf to sunlight shows blue and black spots wherever chlorophyll is present to show a positive starch test.

Conclusion: Green parts of the leaf contain chlorophyll. Hence they carry out photosynthesis and produce starch which turns blue-black when tested with iodine. This experiment proves that sunlight and chloroplast are important for photosynthesis.

Experiment to Demonstrate that Carbon Dioxide is Necessary for Photosynthesis (Moll’s Half leaf experiment): 

Aim: To demonstrate the use of carbon dioxide in photosynthesis.

Materials Required: Potted plant, beaker, KOH solution.

• Take a potted plant and enclose a part of one leaf in a test tube.
• Fill this test tube with some KOH soaked cotton (which absorbs CO 2 ) while the other half of the leaf is exposed to air.
• Pluck the half-covered leaf after a few hours.
• Dip the leaf in iodine solution.

Conclusion: When the two halves of leaf were tested for starch, it was found that only the exposed part of the leaf tested positive for starch. This showed us that CO 2 is required for photosynthesis.

Photosynthesis is the process used by green plants to form glucose molecules by using water, carbon dioxide and chlorophyll molecules. Various scientists performed several experiments to prove the usage of light and carbon dioxide in the formation of food in plants. Scientists like Priestley and Ingenhousz proved that the plants in the presence of light release pure air (oxygen), which is later taken up by other organisms for the purpose of respiration. Later on, Engelmann performed several experiments to give the action spectrum of light and proved that the maximum photosynthesis occurs in the blue and red light of the visible spectrum.

Frequently Asked Questions (FAQs) on Early Experiments to Understand Photosynthesis

Q.1. Which plant was used by Ingenhousz? Ans: Ingenhousz used an aquatic plant Hydrilla to prove plants produce pure air.

Q.2. Which process involves the release of oxygen in plants? Ans: Oxygen is released in plants by the process of the photolysis of water.

Q.3. Which chemical solution was used in Moll’s half leaf experiment? Ans: KOH was used in Moll’s half leaf experiment to prove that carbon dioxide is necessary for photosynthesis purposes.

Q.4. Where does photosynthesis take place? Ans: Photosynthesis takes place in the green part of the plant, which contains chloroplast.

Q.5. Which plant was used by Engelmann to show the action spectrum of the light? Ans: Engelmann used Cladophora to show the action spectrum of light.

Study Mechanism Of Photosynthesis Here

We hope this detailed article on the Early Experiments to Understand Photosynthesis helps you in your preparation. If you get stuck do let us know in the comments section below and we will get back to you at the earliest.

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Mohl's half leaf experiment

Experiment to demonstrate moll’s half-leaf experiment for showing that co 2 , light, chlorophyll and water are necessary requirements for photosynthesis:.

Requirements:

A potted plant, caustic potash, wide- mouthed bottle, iodine, split cork, water

1. De-starch a potted plant by putting it in complete darkness for two days.

2. Fill partly a wide-mouthed bottle with strong solution of caustic potash and fit a split cork on its mouth.

3. Insert about half of the portion of a leaf of the de-starched plant into the bottle through the split cork (Fig. 36).

4. Place the whole apparatus in light after applying grease on the upper portion of split cork, and test the leaf for stach after about 10 hours.

Observations:

Portions of the leaf inside the bottle as well as in between the split cork show negative test for starch indicating the absence of photosynthesis while the portions outside the split cork show positive test for starch indicating the presence of process of photosynthesis in this region.

Negative starch test by the leaf portion present inside the bottle indicates that process of photosynthesis is absent in this region. This portion of leaf is getting all the essential requirements, i.e., light, chlorophyll and water except the CO 2  because the latter is absorbed by the caustic potash. Thus, it can be concluded that CO 2  is necessary for this process.

Precautions:

1. The half leaf kept inside the bottle should not touch the KOH solution. 

2. The apparatus should be kept air tight by applying grease Or baseline. 

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Moll’s half leaf experiment proves that ________ is essential for photosynthesis to take place. a) chlorophyll b) carbon dioxide c)light d)water

• Question Answer
• Molls half leaf experiment pro...

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Moll's half - leaf experiment proves that _________ is essential for photosynthesis to take place. chlorophyll C O 2 light H 2 O

The correct option is b c o 2 in moll's half leaf experiment a destarched plant is used.half part of one of the leaves from the plant is inserted into a corked bottle containing either koh solution or koh palletes. the plant is then placed in sunlight for a few hours. the leaf is then tested for starch with iodine solution. part of the leaf that was inserted in the bottle does not turn blue or test positive for starch. this is because c o 2 present in the bottle was aborbed by koh and the bottle was corked to prevent any entry of air.proving that c o 2 is essential for photosynthesis. so, the correct answer is $co _2$.

A student sets up an experiment on photosynthesis as follows: He takes soda water in a glass tumbler and add chlorophyll extracts into the contents and keeps the tumbler exposed to sunlight hoping that he has provided necessary ingredients for photosynthesis to proceed (viz., CO2, H2O, chlorophyll and light). What do you think what will happen after, say, a few hours of exposure of light?

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How do you prove experimentally that carbon dioxide is necessary for photosynthesis by Mohl’s half leaf experiment? 

How do you prove experimentally that carbon dioxide is necessary for photosynthesis by Mohl’s half leaf experiment? 

List out the materials required and the procedure to be followed to prove that ‘carbon dioxide’ is essential for photosynthesis. 

You know that the factors like CO 2 , Light and Chlorophyll are essential for photosynthesis. 

Write any one of experiment related to the factors essential for photosynthesis.

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To prove-that carbon dioxide is essential for photosynthesis by Mohl’s half leaf experiment.

Apparatus: 

Wide mouthed transparent bottle, KOH solution, potted plant, vertically split cork, Iodine solution. Procedure: Arrange the apparatus as shown in the figure. 

1. Take a healthy potted plant and keep it in the dark for nearly a week for the removal of starch from the leaves

2. A wide mouthed transparent bottle is taken. 

3. Put potassium hydroxide pellets or potassium hydroxide solution (KOH) in the bottle. 

4. This KOH absorbs CO 2 present in the bottle. 

5. Insert splitted cork in the mouth of the bottle. 

6. Insert one of the leaves of destarched plant through a split cork into transparent bottle. 

7. Arrange half of the leaf is inside bottle and the remaining half outside. 

8. Leave the plant in the sunlight for 2-3 hours. 9. After a few hours, test this leaf and other leaf of this plant for starch.

Observation :

1. The part of the leaf outside the bottle turns blue-black because starch is formed in this part due to photosynthesis. 

2. The part of the leaf inside the bottle does not turn blue-black because the carbon dioxide present inside the bottle is absorbed by potassium hydroxide solution. 

3. All the other factors water, sunlight and chlorophyll are available but not CO 2 . Hence starch is not formed in the leaf part which is inside the bottle. 

Result: This experiment proves that CO 2  is necessary for photosynthesis. Precautions: 

1. The part of the leaf kept inside the bottle should not touch potassium hydroxide solution. 

2. The apparatus should be kept air tight by applying grease or vaseline.

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Research Article

An Integrated Approach to Rapid Diagnosis of Tuberculosis and Multidrug Resistance Using Liquid Culture and Molecular Methods in Russia

* E-mail: [email protected]

Affiliations National Mycobacterium Reference Laboratory, Institute of Cell and Molecular Sciences, Queen Mary College, Barts and the London School of Medicine, University of London, London, United Kingdom, Samara Oblast Tuberculosis Dispensary, Samara, Russia

Affiliation National Mycobacterium Reference Laboratory, Institute of Cell and Molecular Sciences, Queen Mary College, Barts and the London School of Medicine, University of London, London, United Kingdom

Affiliation Samara Oblast Tuberculosis Dispensary, Samara, Russia

Affiliation Samara City Tuberculosis Service, Samara, Russia

Affiliations Foundation for Innovative New Diagnostic, Cointrin/Geneva, Switzerland, US Centers for Disease Control and Prevention, Division of TB Elimination, Atlanta, Georgia, United States of America

Affiliation Foundation for Innovative New Diagnostic, Cointrin/Geneva, Switzerland

• Yanina Balabanova, 
• Francis Drobniewski, 
• Vladyslav Nikolayevskyy, 
• Annika Kruuner, 
• Nadezhda Malomanova, 
• Tatyana Simak, 
• Nailya Ilyina, 
• Svetlana Zakharova, 
• Natalya Lebedeva, 

• Published: September 23, 2009
• https://doi.org/10.1371/journal.pone.0007129
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To analyse the feasibility, cost and performance of rapid tuberculosis (TB) molecular and culture systems, in a high multidrug-resistant TB (MDR TB) middle-income region (Samara, Russia) and provide evidence for WHO policy change.

Performance and cost evaluation was conducted to compare the BACTEC™ MGIT™ 960 system for culture and drug susceptibility testing (DST) and molecular systems for TB diagnosis, resistance to isoniazid and rifampin, and MDR TB identification compared to conventional Lowenstein-Jensen culture assays.

698 consecutive patients (2487 sputum samples) with risk factors for drug-resistant tuberculosis were recruited. Overall M. tuberculosis complex culture positivity rates were 31.6% (787/2487) in MGIT and 27.1% (675/2487) in LJ (90.5% and 83.2% for smear-positive specimens). In total, 809 cultures of M. tuberculosis complex were isolated by any method. Median time to detection was 14 days for MGIT and 36 days for LJ (10 and 33 days for smear positive specimens) and indirect DST in MGIT took 9 days compared to 21 days on LJ. There was good concordance between DST on LJ and MGIT (96.8% for rifampin and 95.6% for isoniazid). Both molecular hybridization assay results correlated well with MGIT DST results, although molecular assays generally yielded higher rates of resistance (by approximately 3% for both isoniazid and rifampin).

With effective planning and logistics, the MGIT 960 and molecular based methodologies can be successfully introduced into a reference laboratory setting in a middle incidence country. High rates of MDR TB in the Russian Federation make the introduction of such assays particularly useful.

Citation: Balabanova Y, Drobniewski F, Nikolayevskyy V, Kruuner A, Malomanova N, Simak T, et al. (2009) An Integrated Approach to Rapid Diagnosis of Tuberculosis and Multidrug Resistance Using Liquid Culture and Molecular Methods in Russia. PLoS ONE 4(9): e7129. https://doi.org/10.1371/journal.pone.0007129

Editor: Keertan Dheda, University of Cape Town, South Africa

Received: March 18, 2009; Accepted: August 24, 2009; Published: September 23, 2009

Copyright: © 2009 Balabanova et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: The work was jointly funded by Foundation of Innovative New Diagnostics (FIND), Queen Mary College, University of London and Hain Lifesciences GmbH, Nehren, Germany. Neither Becton Dickenson Diagnostic Systems nor Hain Lifesciences GmbH had a role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Tuberculosis (TB) remains one of the leading causes of morbidity and mortality globally, focused principally, but not exclusively, in the non-industrialized world.

Timely diagnosis and prompt treatment of infectious cases are the key elements of the international effort to combat TB, providing cure of an individual patient and reducing the spread of TB by rendering infectious cases non-infectious.

Multidrug-resistant TB (MDR TB), i.e. resistance to at least isoniazid (Inh) and rifampin (Rif), and extensively drug-resistant TB (XDR TB), i.e. MDR plus resistance to amikacin, kanamycin or capreomycin and a fluoroquinolone, are the most problematic forms of resistance because treatment options are limited and the second-line drugs used for therapy are more toxic, less effective, more expensive, and must be administered for a longer period of time than standard first-line drug therapy [1] .

The highest rates of MDR TB in the world (approximately 10% in new and 25% in re-treatment cases), have been reported from the Baltic region and countries of the former Soviet Union [2] – [11] .

Conventional culture and DST on solid media is a slow process, and in high income, low-incidence countries these systems have been supplemented (or replaced) by automated liquid culture systems such as the Becton Dickinson BACTEC™ MGIT™ 960 system. Decreased time to detection, greater sensitivity than Lowenstein-Jensen (LJ) solid media, comparable sensitivity to the radiometric Bactec 460 system in detecting Mycobacteria in clinical specimens, and good concordance with both LJ and Bactec 460 DST for first-line drugs (FLD) have been demonstrated in several studies [12] – [16] .

Rapid molecular methods, including commercial or in-house DNA hybridisation or amplification methods [17] allow detection of TB and rifampin resistance (and, for some, assays isoniazid resistance as well) in clinical samples within 1–2 days [18] – [25] . Despite the demonstrated advantages, the limited data on the performance, role and value of rapid culture, DST and molecular detection systems, together with concerns of increased cost and contamination rates relative to conventional culture on solid media, have dampened interest and progress in implementing these systems in low to middle income settings. However, this situation is changing in response to the growing MDR TB epidemic and the recent WHO recommendations on the use of liquid culture and DST and line probe assays (LPAs) [26] , [27] .

This study describes the feasibility of introduction, diagnostic accuracy and costs of the MGIT rapid culture system for primary specimens and FLD DST, coupled with rapid molecular systems for TB culture identification and detection of resistance to isoniazid and rifampin in Samara, Russia, a middle income region with a high burden of MDR TB [5] , [9] , [28] . The project was undertaken with the intention (achieved) of producing evidence for the implementation of global health policy changes relating to TB diagnosis by the WHO.

Materials and Methods

Ethics statement.

The study was approved by the Samara Medical University Ethics Committee. The study received a waiver of informed consent because the study used samples that were routinely collected for use in approved routine tests on LJ media. The tests on the MGIT 960 system were performed in parallel with the approved routine tests on LJ media. All aspects related to culture and phenotypic DST were reviewed and approved by the U.S. Centers for Disease Control and Prevention as non-human subjects research which does not require informed consent.

Setting and Design

A programmatic intervention and evaluation was conducted to compare the feasibility, utility and performance characteristics (recovery rates, time-to-detection) of the MGIT culture and DST system (Becton Dickinson, Sparks, MD) and rapid molecular systems to conventional standard reference LJ-based assays in patients at high risk of MDR TB in the central TB laboratory of Samara Region (Category 3 level facility), Russia Federation. The study was preceded by development of an agreed customer support plan that included installation and maintenance of the BACTEC™ machine as well as an uninterrupted supply of reagents needed based on reduced pricing policy offered for Samara. Principles outlined by the Standards for the Reporting of Diagnostic accuracy studies (STARD) for diagnostic accuracy studies were followed.

Prior to implementation into routine practice, MGIT and molecular methods were quality controlled and validated by the Health Protection Agency National Mycobacterium Reference Unit (HPA NMRL) according to the WHO/IUATLD Supranational Reference Laboratory (SRL) proficiency testing criteria [29] using a panel of defined M. tuberculosis cultures provided through the WHO SRL mechanism. Once the laboratory achieved pre-determined minimum standard efficiency levels based on WHO/IUATLD Supranational Reference Laboratory proficiency testing criteria for performance [29] of 80% (E and S), 89% (Inh), and 95% (Rif) in performing MGIT DST, MGIT and LJ culture and FLD DST were performed in parallel and all results were made available to clinicians. All staff was formally trained in bacteriological and molecular methods. Staff performing molecular assays was blinded to culture results and both were blinded to epidemiological data. This study also evaluated the detailed costs associated with the introduction of MGIT for TB culture diagnosis and DST in comparison with conventional methods on solid LJ media.

Patient Population

Patients were enrolled from 8 TB clinics in Samara Oblast that are part of a specialised service which provides diagnostics and treatment for TB patients only. The clinics verify the diagnosis for patients referred by the general health care sector after initial screening with a high suspicion for TB. All confirmed or suspected pulmonary TB patients who were sputum smear-positive and/or at high risk for MDR TB (individuals with prior TB treatment, persistent smear-positivity after 3 months treatment and/or poor clinical improvement, relapse, default, repeated treatment interruptions, contacts of patients with confirmed MDR TB, homeless persons and former prisoners) were included.

Patients were excluded if they were currently receiving TB treatment and smear-converted or remained smear-negative, were known to be infected with an MDR TB strain, or were suspected of having extra-pulmonary TB without pulmonary involvement.

Enrolment commenced in April 2006 and continued through April 2008.

Specimen preparation and primary culture

At least three routine sputum samples were collected from each patient into 50 ml screw-cap centrifuge tubes (Falcon, Becton Dickenson, USA) prior to treatment initiation as well as during treatment as follow-up control samples. Sputum samples were sent daily from the clinics to the laboratory; specimens were stored at 4°C until processed.

Specimens were processed using the NaOH-NALC method [30] employing the Becton Dickinson MycoPrep™ kit as described by the manufacturer. The final concentration of NaOH (1.0% w/v) was determined during the validation phase to maintain contamination rates below 8–10%. Concentrated specimens were stained for the presence of acid-fast bacilli (AFB) according to the Ziehl-Nielsen method [31] .

All processed specimens were immediately inoculated on both MGIT (0.5 ml inoculum) and LJ media (0.2 ml inoculum).

The MGIT 960 was checked daily for positive and negative cultures and LJ cultures were checked at least weekly. Although this standard approach carried an observation bias for time to detection of positive cultures, it followed the accepted practice of periodic visual scanning of LJ slopes reported in all previous published studies of the MGIT and comparable systems and LJ culture. Tubes flagged as positive by the MGIT 960 instrument were examined visually for potential mycobacterial growth and growth was inoculated onto a blood agar plate, subcultured on an LJ slant and MGIT for DST, and an AFB smear was prepared.

Isolate identification

Cultures were identified as M. tuberculosis complex (MTC) according to colony morphology, microscopic appearance, and standard biochemical assays, as specified in the Russian Federal guidelines [31] .

Molecular tests also were used to decrease the time to identification of MTC. Previous studies suggested that liquid culture systems would increase the isolation of non-tuberculous mycobacteria (NTM) as well as M. tuberculosis [32] . Therefore, 327 consecutive mycobacterial cultures initially isolated on the MGIT 960 system were identified to species level using the GenoType® CM assay (Hain Lifesciences GmbH, Nehren, Germany).

First Line drug susceptibility (DST)

The drug concentrations used in the MGIT system were (µg/ml): streptomycin (S): 1.0; isoniazid (Inh): 0.1; rifampin (Rif): 1.0; ethambutol (E): 5.0; pyrazinamide (Z): 100.0 [31] , [33] . DST on LJ was performed according to the absolute concentration method, utilizing the following drug concentrations (µg/ml): S: 10.0; Inh: 1.0; Rif: 40.0; E: 2.0 [31] . Sensitivity to pyrazinamide is not routinely tested on LJ media according to Russian Federal guidelines [31] .

As the majority of isolates were MTC (see Results ), subsequent cultures were identified using an in-house macroarray, as described elsewhere [34] and/or a commercial “line probe” assay system for MTC identification and rifampin and isoniazid resistance (GenoType® MTBDR plus , Hain Lifesciences GmbH, Germany). Both systems employed the same basic principles i.e. polymerase chain reaction (PCR) amplification of relevant regions of genes including the katG and rpoB genes and inhA promoter region, followed by reverse phase hybridisation to probes immobilised on a solid phase membrane.

Cost analysis

All laboratory procedures for both LJ and MGIT culture and DST were broken down into their component parts and a detailed time-and-motion study was conducted [35] – [37] . Total salaries, consumables costs, and capital (including equipment) infrastructure costs, maintenance, administrative and overhead costs of the laboratory, as well as transport costs were included in the final analysis.

Prices were converted into US dollars (USD) for this analysis. International pricings for all relevant laboratory resources and consumables for our study were based on published manufacturer suggested retail prices (MSRP) in developed countries such as the US. For local price analysis, procurement pricings specific to Samara with exceptions to MGIT instrumentation and consumables (for which we used the FIND-Becton Dickinson (BD) negotiated price available in 2006) were used. The usage of equipment, reagents, and laboratory space were quantified as minute used per square meter of space and minutes used. Overhead costs were calculated and allocated based on time-observation data particular to building space and staff utilization for each laboratory procedures included in our cost analysis.

Statistical analysis

All data were obtained from records collected by the clinical and laboratory staff and entered in a password protected stand-alone database to maintain confidentiality.

Statistical analysis was performed using the SPSS version 15 package (SPSS, http://www.spss.com ). The difference between rates among different groups was assessed using chi-test (χ 2 ).

In total, 698 consecutive patients were recruited into the study and 2545 sputum samples were subjected to bacteriological examination on both LJ and MGIT media.

Initially, the MGIT 960 system yielded increased rates of culture contamination but rates were quickly lowered to 3.4% by meticulous adherence to the manufacturer's manual and protocols, and with rapid transport and/or refrigeration of samples. The samples collected from patients were immediately refrigerated and stored at +4 for a maximum time of 48 hours prior to decontamination and culture. All samples from participating study sites were transported in cool bags. Sterility checks of water, buffer and NALC solutions and disposables consumables (such as washes from sputum containers, cryovials and laboratory tubes used) were regularly run using blood agar plates. Negative controls of each batch of MGIT tubes and daily logs of all ready-made solutions were used to monitor any potential manufacturer's contamination. In order for any increased culture speed to be valuable, a rapid molecular identification method was essential to identify culture growth in 1 day; this also permitted TB identification in bacterially-contaminated cultures (data not shown). A proportion of cultures was also spoligotyped to exclude cross-contamination within the laboratory.

Of the first 327 consecutive patients with positive mycobacterial isolates, the applied GenoType® Mycobacterium CM assay demonstrated that the vast majority (96.6%) of isolates were M. tuberculosis complex ( Figure 1 ). Subsequently, molecular methods were used to test all subsequent isolates simply for the presence or absence of MTC. Since very few NTMs were isolated, this paper presents results for MTC only. The overall MTC culture positivity rate for MGIT and LJ was 31.6% and 27.1% respectively (χ 2  =  11.9, p = 0.001); for smear positive specimens it was 90.5% and 83.2% (χ 2  =  8.6, p = 0.003) and for smear negative specimens, 20.4% and 16.4% respectively (χ 2  =  10.7, p = 0.001) ( Table 1 ).

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The overall proportion of the total MTC isolates (number confirmed positive cultures by MGIT or LJ/total number positive cultures by either method) which were positive by MGIT was 97.2% (786/809) compared to 81.1% (656/809) for LJ. Of all culture positive specimens, 99.2% of smear-positive and 95.5% of smear-negative specimens were positive by MGIT while LJ recovery rates were 90.9% for smear-positives and 73.2% for smear-negative specimens. The concordance of results between the two systems was high for isolating MTC (92.7%) ( Table 2 ).

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Among culture positives, the overall median time to detection of M. tuberculosis complex was 14 days and 36 days for MGIT and LJ, respectively. Indirect DST from isolates took an additional 9 days in MGIT and 21 days on LJ. Therefore, providing a rapid molecular identification method is available that takes 1–2 days to perform as in the case of the GenoType® MTBDR plus method and comparable methods such as in house in situ hybridisation methods [34] , [38] the overall turn-around time can be as short as 25 days for MGIT vs approximately 60 days for LJ ( Figure 2 ).

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Comparative phenotypic DST data for both methods ( Table 3 ) on all bacteriologically confirmed TB strains demonstrate approximately 63%, 50%, 27%, 60%, and 10% of the patients were resistant to isoniazid, rifampin, ethambutol, streptomycin, and pyrazinamide, respectively. Approximately 50% of cultures were MDR TB and nearly all rifampin-resistant isolates (98.7% and 100% detected by LJ and MGIT, respectively) were MDR TB. There was good concordance between the results obtained by the LJ and MGIT methods ( Table 4 ) with agreement of 96.8% for rifampin, 95.6% for isoniazid but only 91.9% for ethambutol and for 89.5% for streptomycin.

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The overall Inh, Rif and MDR resistance rates in the population as determined by the in-house macroarray (65.7%, 54.6% and 51.0% respectively) and the Hain methods (66.6%, 54.8% and 53.2% respectively) were comparable.

There was good concordance for isoniazid and rifampin resistance between the commercial and in-house low-density array-based methods (88.5% and 80.7% for macroarray and 87.3% and 77.9% for Hain; 87.6% and 84.9% for macroarray and 84.4% and 82.2% for Hain respectively) compared with the MGIT culture or LJ systems The sensitivity and specificity of both methods when compared to either culture system were high: almost 93% and 94% for detection of isoniazid and approximately 87% and 94% for detection of rifampicin resistance against the MGIT system; approximately 92% and 93% for isoniazid and 90% and 93% for rifampicin against the LJ system respectively ( Tables 5 – 6 ).

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Compared to the LJ method, MGIT culture was consistently more expensive than LJ regardless of pricing levels but the difference was small ( Table 7 ). However, the FIND-BD pricing agreement brings about a 40% reduction in overall costs for screening one specimen for full first line DST with MGIT at $32 as compared to $56 for full catalogue pricing. LJ methodology costs ranged from $17 to $20 in Samara and international prices; if only isoniazid and rifampin resistance was tested the equivalent costs for Samara and internationally were $13 and $15 for LJ and $17 and $28 for the MGIT system.

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The cost of performing the in-house method (macroarray) in Samara was calculated based on local wages and overhead costs: the overall average unit cost of the macroarray test was at about $15 per specimen. The total chemical and reagent components of the test was between $5–6 per specimen ( Table 6 ). The test-strips used for this method were produced at the HPA MRU, London; more detailed assessment of the total assay costs when produced in Russia is needed and is subject of another on-going study.

The costs of the GenoType® MTBDR plus assay were not assessed within this project.

This study describes the performance characteristics of rapid liquid culture (MGIT 960) and molecular assays for the identification of MTC, rifampin, isoniazid and MDR TB as well as costs of the MGIT 960 system when introduced into Samara, a region within the Russian Federation with high rates of drug resistance and MDR TB [11] .

The MGIT 960 method was quickly taken up by the staff and successfully introduced into practice in an escalating manner from primary culture to DST. An initially high contamination rate was lowered to 3.4% within a month of initiation of the project by meticulous adherence to manufacturer's instructions, use of standard protocols as well as a well-developed system of rapid sample collection and transport logistics. Coupled with high recovery rates, it demonstrates that decontamination procedure was not overly harsh and permitted adequate growth of mycobacteria while ensuring low contamination rates. A preliminary analysis presented here found that over 96% of positive cultures were M. tuberculosis complex, suggesting that frequent NTM isolation (as reported elsewhere [32] ) was unlikely to be a significant problem in this study population. This was probably due to the high proportion of smear-positivity and drug resistant TB among enrolled patients, lower HIV-positivity rates compared to African countries, and dominance of the Beijing family TB strains in the area. This strain family has been actively transmitted in the area and has a strong association with drug resistance [5] . Subsequent culture growth was identified using a second commercial rapid identification system and a non-commercial in house system for MTC, isoniazid and rifampin resistance which both employed the same principle of PCR amplification coupled with a reverse phase hybridisation detection system.

As reported in high-resource, low-TB incidence settings, a greater proportion of positive cultures from primary specimens grew in the MGIT system primarily due to increased culture sensitivity for smear-negative specimens.

The median time for culture isolation was significantly faster for the MGIT 960 compared to LJ at 14 days versus 36 days for all specimens in agreement with other international studies mainly from low incidence [32] , [39] – [42] .

The proportion of drug resistance was very high in the studied population – almost half of the isolates were MDR TB. Mono –resistance to rifampicin was very rarely seen and nearly all rifampin-resistant isolates were MDR TB suggesting that rifampin resistance may serve as a reliable surrogate marker for MDR TB in this population. The median time to obtain DST results from positive cultures was faster with the MGIT system at 9 versus 21 days for LJ based methods in line with previously published works mainly from countries with lower levels of drug resistance [43] . Therefore introduction of the MGIT method can significantly decrease the overall turn-around time to 25 days comparable to data reported elsewhere [44] ). However, within the project framework the turn-around-time for positive cultures and DST ranged from 13 to 87 days with a median of 38 days for the MGIT. Delays occurred due to logistical problems during the introduction of the MGIT system into routine use (e.g. reagents supply), training of additional personnel, contamination and delays between receiving a culture and subculturing for DST. One of the advantages of using the MGIT system was an opportunity to reliably determine sensitivity to pyrazinamide. This test is not routinely performed on LJ media in Russia due to the lack of standard protocols and variable standardization recommended on the national level.Although the MGIT system generated all FLD DST results more rapidly than the LJ methodology, the molecular methods provided results for isoniazid and rifampin resistance within one day. Another study performed in the same setting of the Samara Regional Tuberculosis Laboratory presented evidence for efficient use of molecular assays (GenoType® MTBDRplus) directly on smear-positive sputum samples [45] . The current study demonstrated that concordance of the commercial and in-house molecular methods for isoniazid and rifampin resistance was high, with very close but not complete agreement for isoniazid and rifampin resistance between the molecular and MGIT defined DST results. These methods could be implemented as an initial screen for MDR TB (directly on smear-positive samples or on mycobacterial cultures isolated from smear-negative samples), permitting the institution of infection control measures at an earlier stage, as well as more rapid provision of appropriate treatment in line with recent WHO recommendations that were developed with the support of the Samara project data [26] . The presence of mutations indicating resistance could be used as an indicator for simultaneously initiating first- and second-line resistance testing in MGIT, which could significantly reduce the delay in administering an appropriate drug regimen to an MDR TB (or XDR TB) case.

The economic analysis demonstrated that although the MGIT culture system was slightly more expensive than the LJ method ($12 versus $11 respectively), it would permit earlier diagnosis of TB and prompt treatment initiation (a reduction in median culture time of 22 days).

Similarly MGIT FLD DST was more expensive than LJ FLD DST ($56 versus $20 using international prices) but the difference allowed a significant decrease in diagnostic turn-around time resulting in earlier identification of drug resistance, including MDR TB, especially when FLD and SLD DST are set up simultaneously for isolates which were diagnosed as having mutations to rifampicin and isoniazid by molecular methods. Coupled with molecular systems for rapid identification and drug resistance detection, this would have a significant impact on a timely administration of an adequate treatment regimen and potentially improve treatment outcomes. Administration of timely therapy will render an individual non-infectious and interrupt transmission; molecular methods and/or MGIT based DST identify the many patients who have MDR TB (who will not be rendered non-infectious by standard TB therapy and so will continue to transmit MDR TB) and culture –based phenotypic methods are the only way of reliably identifying the antimicrobials that are able to render MDR TB individuals non-infectious.

This demonstration project provided much of the evidence underpinning the diagnostic policy changes relating to bacteriological culture adopted by the WHO in 2007-8. Currently WHO recommend the routine use of TB liquid culture and DST even in resource-limited settings to improve diagnosis of TB in general, MDR TB and smear-negative pulmonary TB including application of a rapid method of species identification [27] . The higher cost of the automated liquid culture media systems is currently being addressed by the manufacturers by introducing changes into the pricing policy for the public sector in lower income countries. This project showed that it is possible to successfully introduce this technology into resource-constrained settings but that to achieve satisfactory implementation and performance of the MGIT system (which is more prone to bacterial contamination due to the greater sensitivity of liquid media for culture of mycobacteria as well as other microorganisms compared with solid culture and for DST which is more complex to perform that using solid culture) key issues needed to be resolved. These include: (1) availability of appropriate Category 3 level laboratory infrastructure including an agreed maintenance plan for the BACTEC system; (2) repetitive on-site training of laboratory personnel in MGIT methodology (using detailed SOPs and the system manual) and molecular methods to create a multi-skilled cadre of staff; (3) initial expert observation of the performance and implementation of internal and external quality control of laboratory work; (4) development of effective logistics for timely collection, storage and transport of fresh sputum samples to the laboratory as well as the reporting of results; (5) the creation of algorithms for laboratory work flow and computerized laboratory record keeping; (6) timely maintenance of equipment and ensuring a safe continuous supply of reagents by establishing a commercial contract with a manufacturer and (7) introduction of a robust stock control system.

For these diagnostic culture systems to have a maximum therapeutic impact there must be rapid identification of cultures with the ability to analyse first and second-line DST phenotypically when molecular tests demonstrate the presence of mutations encoding rifampin (and possibly isoniazid) resistance in the original patient specimen or the resulting culture. This will significantly reduce the time between sputum collection and full susceptibility testing for MDR TB cases. Addressing timeliness in technological improvement should go in tandem with minimizing organizational delay. Clinicians need to make prompt therapeutic changes following rapid DST analysis.

With effective planning and logistics, an adequate decontamination protocol and careful training, the MGIT 960 and molecular-based methodologies can be successfully introduced into a reference laboratory setting in a middle TB incidence country. The high rates of MDR TB in the Russian Federation make the introduction of such assays particularly useful and are likely to translate to other settings with a high level of drug resistance or where the additional speed of diagnosis and increased diagnostic sensitivity are of value as in HIV associated tuberculosis.

Acknowledgments

The project was a close collaboration between the Samara TB service, the UK Health Protection Agency and Barts and the London Medical School, University of London, Hain Lifescience Gmbh and the Foundation for Innovative New Diagnostics (FIND).

We would like to thank Dr Olga Kurkina for help with participants' recruitment, Mrs Marina Korobova for performing a proportion of bacteriological work, Ms Olga Ignat'yeva and Ms Svetlana Mironova for performing molecular work on a proportion of isolates.

Author Contributions

Conceived and designed the experiments: YB FD VN AK NM SZ NL HLA RO HS IF. Performed the experiments: YB FD VN AK NM TS NI SZ NL HLA HS. Analyzed the data: YB FD VN TS RO HS AS. Contributed reagents/materials/analysis tools: YB VN AK NM HLA RO HS IF. Wrote the paper: YB FD HLA RO.

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• v.7(1); 2006

The Directly Observed Therapy Short-Course (DOTS) strategy in Samara Oblast, Russian Federation

Y balabanova.

1 HPA Mycobacterium Reference Unit, Clinical TB and HIV Group, St Bartholomew and Queen Mary School of Medicine, 2 Newark street, London E1 2AT, UK

3 Samara City Tuberculosis Dispensary N1, Pionerskaya street, Samara, 443001, Russia

F Drobniewski

2 Samara Oblast Tuberculosis Dispensary, Samara, 154 Novo-Sadovaya Street, 443068, Russia

S Zakharova

V nikolayevskyy.

4 Center for Health Management, Tanaka Business School, Imperial College London, South Kensington campus, London SW7 2AZ, UK

5 Department of Public Health and Policy, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK

This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The World Health Organisation (WHO) defines Russia as one of the 22 highest-burden countries for tuberculosis (TB). The WHO Directly Observed Treatment Short Course (DOTS) strategy employing a standardised treatment for 6 months produces the highest cure rates for drug sensitive TB. The Russian TB service traditionally employed individualised treatment.

The purpose of this study was to implement a DOTS programme in the civilian and prison sectors of Samara Region of Russia, describe the clinical features and outcomes of recruited patients, determine the proportion of individuals in the cohorts who were infected with drug resistant TB, the degree to which resistance was attributed to the Beijing TB strain family and establish risk factors for drug resistance.

prospective study

2,099 patients were recruited overall. Treatment outcomes were analysed for patients recruited up to the third quarter of 2003 (n = 920). 75.3% of patients were successfully treated. Unsuccessful outcomes occurred in 7.3% of cases; 3.6% of patients died during treatment, with a significantly higher proportion of smear-positive cases dying compared to smear-negative cases. 14.0% were lost and transferred out. A high proportion of new cases (948 sequential culture-proven TB cases) had tuberculosis that was resistant to first-line drugs; (24.9% isoniazid resistant; 20.3% rifampicin resistant; 17.3% multidrug resistant tuberculosis). Molecular epidemiological analysis demonstrated that half of all isolated strains (50.7%; 375/740) belonged to the Beijing family. Drug resistance including MDR TB was strongly associated with infection with the Beijing strain (for MDR TB, 35.2% in Beijing strains versus 9.5% in non-Beijing strains, OR-5.2. Risk factors for multidrug resistant tuberculosis were: being a prisoner (OR 4.4), having a relapse of tuberculosis (OR 3.5), being infected with a Beijing family TB strain (OR 6.5) and having an unsuccessful outcome from treatment (OR 5.0).

The implementation of DOTS in Samara, Russia, was feasible and successful. Drug resistant tuberculosis rates in new cases were high and challenge successful outcomes from a conventional DOTS programme alone.

Since the 1990s the World Health Organization Directly Observed Therapy Short Course (DOTS) management strategy has become the internationally recommended approach for tuberculosis (TB) control programmes [ 1 - 3 ]. By the beginning of the new Millennium, 149 countries in the world had adopted the DOTS strategy to varying degrees and important measures of DOTS success (case detection and treatment success) were included in the Millennium Development Goals framework [ 4 ]. In the former Soviet Union (FSU) only a limited number of WHO DOTS implementation programmes exist and currently countries of the FSU report the lowest case detection rates (22%) with 9% of cases failing treatment and a death rate of 7% during treatment [ 4 ]. WHO has acknowledged that until TB is controlled in Africa and Eastern Europe, this disease will remain of major world-wide concern; current analysis indicates that it is unlikely that the Millennium Development Targets for TB will be met in these regions [ 4 ].

Russia is one of 22 TB high-burden countries as defined by the WHO [ 5 , 6 ]. Russia has a highly-specialised tuberculosis health care system with a large organisationally-vertical network of specialized institutes, dispensaries, hospitals, outpatient clinics, sanatoria and rural feldsher points. Case detection is based largely on the presence of radiological abnormalities on chest X-rays with or without bacteriological confirmation detected through a national policy of compulsory annual fluorographic population screening [ 7 - 9 ]. In contrast to the WHO recommended tuberculosis control DOTS strategy, which favour minimising hospital stays, clinical guidelines and health system financing incentives, TB patients in Russia experience frequent and lengthy hospitalisations, and historically have received individualised treatment regimens with doses of the main first line drugs and duration of chemotherapy varying from internationally accepted standard treatment regimens. The system also included prolonged periods of follow-up and repetitive courses of anti-relapse therapy [ 10 , 11 ].

The rationale for implementing the DOTS strategy in Russia is to establish cost-effective tuberculosis control by reducing unnecessary care costs due to lengthy hospitalisations, while improving cure rates and reducing the development of drug resistant TB [ 3 , 7 , 12 - 14 ].

In 2002, with assistance from the UK Department for International Development, a TB control programme that adhered to internationally accepted norms and standards was launched by the regional Ministry of Health. We have reported elsewhere on the considerable body of research undertaken in Samara that explores the epidemiological profile, the health care system structures and processes, and public health challenges being faced by the oblast [ 7 , 9 - 12 ], [ 15 - 23 ]

This paper describes the clinical features and outcomes of patients recruited to a DOTS programme which was implemented in civilian and prison sectors in Samara Oblast.

At the initial stage of implementation of DOTS a standard protocol was agreed with the Regional Ministry of Health. This was followed by extensive training of medical doctors and TB nurses with the involvement of WHO and experts from Russian Federal TB Institutes. Two project medical co-ordinators based in Samara were appointed to oversee implementation which was rolled out in three phases.

Under phase one, initiated in April 2002, patient recruitment commenced at two pilot TB dispensaries in Samara City and at two TB prison colonies (one, an inpatient prison facility used for initial therapy, the second an outpatient facility where continuation of therapy occurred) that looked after all prisoners with TB in the oblast. Recruitment was expanded in January 2003, under phase two, to all TB facilities in Samara city (five dispensaries and three TB hospitals) and to the neighbouring city Togliatti. Under phase three, a further rollout occurred in January 2004 to the rural district of Krasny Yar. We report results through all three phases and include patients recruited up to the third quarter of 2004.

Patients were recruited into standard WHO categories (Table ​ (Table1). 1 ). In 2002, initially only new cases were recruited (category I and III). From April 2003, recruitment was extended to include relapse cases (category II). Because of the prevalence of drug resistance and concerns that resistance profiles would be further amplified [ 23 - 25 ] chronic cases were ineligible for recruitment.

WHO treatment categories and outcome definitions [28]

* Treatment success is defined as the sum of patients cured and those who have completed treatment.

Given implementation of the internationally supported programme ceased in third quarter 2004, clinical outcomes presented are until the third quarter of 2003. Outcomes for patients recruited subsequently were registered within the newly adopted Russian national system which continued following this programme [ 26 , 27 ].

Standard TB control treatment outcomes were recorded (Table ​ (Table1). 1 ). Treatment success under the DOTS strategy was determined by cures and treatment completions and unsuccessful treatment included patients who failed and defaulted [ 28 ].

A modified feature of the programme was introduced where patients registered initially under the DOTS cohort could be transferred to an "individual treatment regimen", an approach that reflected the Russian legacy of individualised approaches to treatment. According to the prevailing views of Samara phthisiatrists not only patients who were diagnosed with MDRTB but also some severely ill patients or those with severe co-morbidities or perceived adverse reactions would be removed from the DOTS programme and managed within the regional TB programme using an individualised approach in line withy earlier Russian traditions. Cases, following recruitment, which were subsequently determined to have MDR TB, were transferred out to an individually-tailored MDRTB drug regimen. A further feature of the modification of the DOTS programme was the continuation of the intensive phase of treatment beyond two months (for one more month) despite patients becoming smear-negative in the end of the second month of therapy intensive phase. This was done, in accordance with Russian traditions, where extensive radiological changes were present.

Standard technical approaches to documentation and diagnostic/treatment protocols were employed[ 28 ]. Sputum collection was performed at recommended intervals. During the intensive phase of therapy ethambutol was administered instead of streptomycin because a previous drug resistance survey had documented very high rates of primary resistance to streptomycin [ 9 , 23 ].

For all patients smear microscopy and culture was performed at recommended intervals. Smear microscopy and culture were performed using standard Ziehl-Neilsen microscopy and culture on Lowenstein-Jensen media. All positive isolates were tested for drug susceptibility to isoniazid, rifampicin, ethambutol and streptomycin. Quality-assured drug susceptibility testing (DST) was performed at three civil and one prison site using an absolute concentration method on Lowenstein-Jensen media. DST was assured by a period of training by staff from the WHO Supranational Reference Laboratory (SRL) in London (Health Protection Agency MRU) and in Samara. A blinded analysis of a test panel of TB cultures was performed. A proportion (10%) were retested by the SRL in London.

DNA was extracted and Beijing family strains were analysed in London and Samara by detection of the IS6110 insertions in the dnaA-dnaN intergenic region on a proportion of sequential isolates (n = 740).

Direct supervision of treatment adherence was completed by TB nurses at TB hospitals and dispensaries with out-patients receiving treatment daily or three times weekly. Upon release, ex-prisoners completed their treatment upon transfer of their care to the civilian service.

Medical co-ordinators performed regular visits to all participating DOTS sites to support implementation, ensure recruitment was maintained, and review documentation and adherence to the protocol. Over-arching project management group meetings which included all clinical stakeholders and the project directors from each DOTS site occurred on a monthly basis.

Socially disadvantaged patients were identified by a responsible physician at each dispensary and offered additional support to encourage treatment adherence with weekly food packages at a cost of 100 Russian Roubles (3 Euros) per person per week.

Data were entered and stored into a password protected database. The statistical analysis was performed using Excel and SPSS 12. Proportions with 95% confidence intervals (CI), relative risks (RR), odds ratios (OR), and χ 2 test are used for comparison of categorical variables.

The study was approved by the Samara Regional Ethics Committee.

2,099 patients were recruited from 1 st April 2002 to 30 th September 2004, including 1,971 individuals with pulmonary tuberculosis (93.9%) and 128 patients with extrapulmonary disease (6.1%); 1,684 of recruits were men (80.2%) and 415 (19.8%) women. 640 patients were recruited in the prison sector and 1459 were civilian TB patients.

One third (33.1%; 694/2,099) of recruited patients were WHO category I and 24.3% (162/694) of these were smear-negative cases with extensive parenchymal involvement; 58.8% (1,234/2,099) of cases were WHO category III patients. Recruitment into WHO category II was limited to relapse cases only and 171 patients (8.1%) were recruited.

The mean age of patients was 38.5 years (95%CI 37.9–39.1 years; range: 16–90 years) with prisoners being significantly younger than civilians (mean age 30.9 years; 95%CI 30.2–31.6 years versus mean age 41.9 years; 95%CI 41.1–42.7 years). Female patients were older than male patients (mean age of men was 38.0 (95%CI 37.4–38.6) years and mean age of women with TB was 40.4 (95%CI 38.8–42.0) years).

Details of bacteriologically (smear and/or culture) confirmed cases are shown in Table ​ Table2. 2 . Table ​ Table3 3 shows differences between the infectious status of civilian and prison populations with TB where civilians were more likely to have infectious disease whether determined by smear status or culture status. Overall the rate of laboratory diagnosed TB cases was slightly higher in civilian patients than prisoners.

Proportion of bacteriologically confirmed new and relapse pulmonary cases (II quarter 2002 – III quarter 2004)

*a proportion of patients could not expectorate a sputum sample of a quality that would be suitable for culturing

Difference in infectious status between civilians and prisoners

* statistically significant at p < 0.05

Cultures from 948 sequentially new and 94 relapse cases were isolated and tested for susceptibility to first-line drugs. Of the new cases, 24.9% (236/948) new cases had isolates resistant to isoniazid, 20.3% (192/948) new cases had isolates resistance to rifampicin, and 17.3% (164/948) had MDRTB (vs 34.0% (32/94) of relapse cases being MDR (OR-2.5; 95%CI 1.6–3.9). Table ​ Table3 3 and Figure ​ Figure1 1 show the differences between civilian and prison patients.

Rates of first-line drug resistance among civil and prison patients.

Molecular epidemiological analysis demonstrated that a half of all isolated strains (50.7%; 375/740) belonged to the Beijing family. Of note, seven isolates (0.9%) were mixed strains. The prevalence of the Beijing strain (60.9%; 117/192) among prisoners was significantly higher (OR-1.7; 95% CI 1.2–2.4) than in civilians (47.1%; 258/548) confirming earlier research findings in a drug resistance survey in the same region in the preceding year [ 19 ].

For 709 isolates data on both drug resistance and strain type were available (31 isolates were non-viable or were contaminated and DST could not be performed). Drug resistance including MDR TB was strongly associated with being infected with the Beijing strain (for MDR TB 35.2% in Beijing strains versus 9.5% in non-Beijing strains, OR-5.2 (3.4–7.9) (Table ​ (Table5) 5 ) confirming earlier research in a different population of patients treated under the Russian system in the same region[ 19 ].

Comparison of first-line resistance levels in Beijing compared to non-Beijing strains (n = 709^)

* statistically significant difference at p < 0.001

^ both epidemiological and drug resistance results were available for 709/1042 (68.0%) cultures; 31 cultures were non viable or contaminated; cultures were drawn from all dispensaries and prison facilities in Samara City.

Difference in resistance rates between civil and prison patients

*statistically significant at p < 0.05

Multivariate analysis suggests that being a prisoner (OR – 4.4; 95%CI 2.7–7.1), having a relapse of TB (OR-3.5; 95%CI 1.7–7.1), being infected with the Beijing family strains (OR-6.5; 95%CI 4.0–10.5) and having unsuccessful outcome of treatment (OR-5.0; 95%CI 1.1–22.7) were risk factors for MDR TB.

During the course of treatment the majority (97.7%; 284/290) of smear-positive new cases converted by the end of the intensive phase of treatment.

Treatment outcomes among new cases confirmed by culture are shown in Table ​ Table6. 6 . Because recruitment of relapses was initiated at a later stage, the number of these is small. Overall 85.4% (786/920) of newly diagnosed and recruited patients were treated according to the WHO protocol. Nearly fifteen percent (134/920) of patients were transferred out of the DOTS clinical protocol and this included patients transferred to individual regimens because MDRTB (17.3% of all new cases were MDR and 34.0% of all relapse cases) or extensive radiological abnormalities, adverse drug reactions, or co-morbidities). MDR TB patients were removed from the programme according to DOTS project criteria and further treated with tailored schemes using second-line drugs. More smear positive patients were transferred out than smear-negative cases (22.4% versus 11.6%; OR-2.2; 95%CI 1.5–3.2). In total 75.3% (592/786) of patients were successfully treated and in 7.3% (57/786) treatment failed or patients defaulted. The odds of failing treatment or defaulting were higher in smear positive patients (OR – 10.6; 95% CI 3.4–32.8).

Cohort outcomes for new cases confirmed by culture

*a proportion of patients were excluded from cohort (transferred to a different regimen due to MDR TB, extensive radiological abnormalities, drug adverse reactions, severe accompanying pathology)

** N of patients remained in the cohort (column "total") is taken as a denominator

There was no statistically significant difference in treatment outcomes between male and female patients.

The rates of unsuccessful treatment was higher among civilians compared to prisoners (OR-4.5; 95%CI 2.1–10.0)

Twenty-eight patients (3.6 %; 28/786) died during the course of treatment with a significantly higher proportion (11.3%) of smear-positive cases dying versus smear-negative (OR -12.5; 95%CI 5.0–31.3).

Discussion and conclusion

The WHO have argued that the introduction of DOTS cohort treatment strategies improves case detection and treatment and leads to a reduction in TB prevalence and death rates by cutting the duration of illness and case fatality.

Two examples from middle and high incidence countries (Peru and China) support this view. In Peru, the incidence rate of pulmonary TB has decreased annually by 6% after the nationwide implementation of DOTS[ 29 ]. In 13 provinces of China that implemented DOTS, the prevalence rate of culture-positive TB was cut by 30% between 1990 and 2000 [ 30 ]

The introduction of DOTS resulted in profound changes to the delivery of clinical care within the Samara TB Service. Although a direct observation component had, broadly. been present within the old system through lengthy hospitalisation periods, the strict adherence of physicians to standard regimens, the emphasis on laboratory diagnosis, and a robust system of recording and reporting of cohorts were new [ 7 ].

Similarly fewer than 70% of patients with TB were cured or completed treatment in Samara compared to 75.3% in the cohort groups. This is in keeping with the cure rates reported for DOTS programmes internationally (Table ​ (Table7) 7 ) and the global treatment success rate under DOTS has been high since the first observed cohort in 1994 (77%)[ 4 ].

Comparison of Samara DOTS cohort with overall treatment success and outcome for global DOTS cohorts in 2002

Established markets include member states of the EU, North America, Australia, New Zealand and Japan.

The relatively high failure rates noted elsewhere in Eastern Europe, (9% of cases failed treatment and 7% died during treatment) are believed to be associated with high rates of multidrug resistance (which in itself is an indicator of a programme with low cure rates previously). In Samara, prior to the introduction of the DOTS cohort strategy we established that drug resistance was high in both new (approx 20%) and chronic cases in Samara [ 9 , 17 , 23 ].

Dye et al [ 4 ] further established that the prevalence will decrease sooner if case detection by DOTS programs (and hence the quality of treatment) can be improved more quickly, thus reducing the burden of illness during this period in future years. The DOTS programmes emphasise the importance of bacteriological confirmation. [ 7 ]Prior to the establishment of the cohort, there were more than 1.5 million flurographic examinations of the general population for early diagnosis of TB reflecting the national policy of fluorography screening of the population for TB for early diagnosis. We have reported on the subjective nature of radiological examination elsewhere [ 31 ] and emphasised the need for bacteriological confirmation of the diagnosis in line with international standards.

Previously, less than one-third (30.1%) of cases were bacteriologically confirmed (Coker et al, 2003 IUATLD) compared to the DOTS cohort where 49.6% of all cases (and 57.6% in civilian cases) were bacteriologically confirmed. [ 10 , 11 ]Overall the proportion of cases which had a bacteriological confirmation of the diagnosis was similar to rates reported from other regions of Russia [ 4 ];.

[ 31 ]Although the laboratory component of the TB service in Samara Oblast has been extensively upgraded and improved with prison and civil laboratory services working to these improved standards, maintenance and further quality improvement remains a priority. Without appropriate laboratory support, over-diagnosis of tuberculosis remains a possibility, resulting in unnecessary treatment and side-effects without benefit, and compounding service inefficiencies [ 13 ].

Relatively low default rates occurred with implementation of DOTS in Samara. This may be, in part, attributable to a programme of externally financed social support. This component was discontinued after external funding ceased, and it remains to be seen whether adherence rates will suffer. Of note, substance abuse, alcoholism, poverty and unemployment are common amongst patients with TB in Samara Oblast, co-factors likely to influence treatment adherence [ 22 ]. The sustainable success of DOTS in Russia is likely to be dependent on how care and support for these social and behavioural factors are integrated into TB care systems.

Effective responses in support of TB control demand political commitment and investment from local and federal budgets into non-medical support to patients and their families. However, few integrated social support systems for tuberculosis patients currently exist, and current laws and regulations have the potential to ensure that health and social care budgets remain disconnected from each other and from need [ 32 , 33 ]. Consequently, to compensate for inadequate social support systems for tuberculosis patients, providers use sophisticated practices to ensure lengthy admissions in the winter months – a response to social rather than medical need [ 32 , 33 ]. Whilst the two recent decrees on TB control issued in 2003 (#109 and #50) [ 26 , 27 ] support convergence of Russian TB control practices with WHO's DOTS strategy (with some specific differences reflecting Russia's clinical legacy), the sustainability of reforms needed to ensure cost-effective implementation such that DOTS implementation is allied to structural reform remains uncertain.

The rate of successful treatment (75.3% overall and 79.0% in civil sector) though below the 85% WHO target, was higher than reported from other several DOTS pilot regions in the former Soviet Union (68.1% according to the meta-analysis performed by Faustini et al, 2005 [ 25 ]). The zero mortality among prisoners may be misleading: several patients died after data censoring. Furthermore, policy that very severely ill patients are released from prison for treatment in the civilian sector means that deaths of these ex-prisoners are recorded as civilian deaths.

The high prevalence of drug resistance and the frequency of the Beijing strain family (previously shown to be associated with drug resistance) [ 19 ] remains a major clinical and public health challenge. Extremely high rates of drug resistance among prisoners despite significantly lower default rates in prison likely reflects on-going transmission of resistant strains. Rapid isolation of MDR TB cases, good co-ordination between the prison and civilian TB services and enhancement of infection control and treatment are needed to prevent further nosocomial and institutional spread of MDR TB and would increase the success of the current TB programme. This issue is likely to become considerably more of a problem as the emergent epidemic of HIV in Samara matures

Although the DOTS strategy does not include specific therapy for multi-drug resistant cases its effective implementation reduces the occurrence and further transmission of resistant strains [34]. However, in regions such as Samara with very high rates of MDR TB it is essential to ensure the availability of appropriate and timely diagnosis and treatment of existing cases as well as preventing the development of new ones. Rapid drug susceptibility techniques, with appropriate treatment to be tailored to circumstance may be necessary. Cost-effectiveness analysis of rapid methods in the post-Soviet context is required to inform investment and policy changes.

Abbreviations

CI – Confidence Interval

DOTS – Directly Observed Therapy Short-Course

MDR TB – multi-drug resistant tuberculosis

OR – Odds ratio

RR – Risk ratio

SRL – Supranational Reference Laboratory

TB – tuberculosis

WHO – World Health Organization

Competing interests

The author(s) declare that they have no competing interests

Authors' contributions

YB participated in the design of the study, its coordination, acquisition of data, statistical analysis and drafted the manuscript; RC participated in acquisition of funding, design of the study, its supervision and drafted the manuscript; IF participated in administration of the study, its design and revision of the manuscript; SZ participated in design and coordination of the study, data acquisition and manuscript revision; VN carried out laboratory work and revised the manuscript, RA participated in acquisition of funding, design of the study, its supervision and revision of the manuscript, FD participated in acquisition of funding, design of the study, its supervision and gave final approval of the version to be published. All authors read and approved the final manuscript.

Acknowledgements

The UK Department for International Development (DFID) funded this study, but the views and opinions expressed are those of the authors alone.

We would like to thank all doctors and nurses who took part in this study.

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Mohl’s half leaf experiment proved that

The correct option is b co 2 is essential for photosynthesis mohl’s half leaf experiment was conducted to demonstrate the importance of co 2 in photosynthesis. the part of the leaf exposed to koh did not synthesise food through photosynthesis since koh absorbed all the co 2 from the immediate surroundings of the leaf. jan ingenhousz’s aquatic plant experiment proved that sunlight is essential for photosynthesis. jan bapista van helmont’s willow tree experiment was used to prove that water is essential for photosynthesis. variegated leaf experiment was used to prove that chlorophyll is essential for photosynthesis..

Lysanderhöhe (Am Trakt Mennonite Settlement, Samara Oblast, Russia)

Lysanderhöhe was a village of the Am Trakt Mennonite settlement in the province of Samara , Russia , founded in 1864, consisting in 1897 of 22 farms, with a population of 119. A part of the population joined the trek to Turkestan in 1891, while others immigrated to America. In 1914 the population was 146. Franz Bartsch , the author of Unser Auszug nach Mittelasien, was the teacher of the village school. After the Revolution of 1917 the cultural and economic level of the village began to decline. Some of the inhabitants went to Canada and others were exiled. Little is known about the fate of those who remained and the later status of the village.

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Hege, Christian and Christian Neff. Mennonitisches Lexikon , 4 vols. Frankfurt & Weierhof: Hege; Karlsruhe: Schneider, 1913-1967: v. II, 709.

Surukin, W. E. und Johannes J. Dyck. Am Trakt, Eine Mennonitische Kolonie im Mittleren Wolgagebiet .North Kildonan, Man., 1948.

Cite This Article

Krahn, Cornelius. "Lysanderhöhe (Am Trakt Mennonite Settlement, Samara Oblast, Russia)." Global Anabaptist Mennonite Encyclopedia Online . 1957. Web. 19 Aug 2024. https://gameo.org/index.php?title=Lysanderh%C3%B6he_(Am_Trakt_Mennonite_Settlement,_Samara_Oblast,_Russia)&oldid=144294 .

Krahn, Cornelius. (1957). Lysanderhöhe (Am Trakt Mennonite Settlement, Samara Oblast, Russia). Global Anabaptist Mennonite Encyclopedia Online . Retrieved 19 August 2024, from https://gameo.org/index.php?title=Lysanderh%C3%B6he_(Am_Trakt_Mennonite_Settlement,_Samara_Oblast,_Russia)&oldid=144294 .

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